Role of WT1 in mixed phenotype acute leukemia

WT1在混合表型急性白血病中的作用

基本信息

批准号：
10457433
负责人：
Lindsey Montefiori
金额：
$ 2.04万
依托单位：
ST. JUDE CHILDREN'S RESEARCH HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-01 至 2022-09-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10457433
关键词：
Acute Lymphocytic Leukemia Acute leukemia Address Apoptosis Binding Biological Assay Biology Biotin Blood Cells C-terminal CD34 gene CRISPR/Cas technology Cancer Model Cell Separation Cell model Cells Child Childhood Childhood Leukemia Chromatin Chromatin Structure Complement Complex DNA Binding DNA Binding Domain DNA Methylation Dependence Development Disease Epigenetic Process Event Exhibits Exons Fellowship Flow Cytometry Frequencies Future Gene Expression Gene Expression Profiling Gene Expression Regulation Genes Genetic Genetic Enhancement Genetic Models Genetic Transcription Genomics Goals Guide RNA Hematopoiesis Hematopoietic Hematopoietic stem cells Human Impairment In Vitro Knock-out Knowledge Label Lentivirus Leukemic Cell Lymphoid Lymphoid Cell Malignant - descriptor Malignant Childhood Neoplasm Maps Mediating Methodology Modeling Molecular Molecular Target Mutate Mutation Myelogenous Myeloid Cells Pathway interactions Patients Phenotype Play Population Prevalence Process Prognosis Proteins Proteomics Recurrence Regimen Regulation Regulator Genes Research Research Proposals Research Training Resources Role Sampling Surface Antigens Terminator Codon Testing Therapeutic Therapeutic Intervention Transcriptional Regulation Transplantation Treatment outcome Umbilical Cord Blood Uncertainty WT1 gene Zinc Fingers base bisulfite sequencing cell type chromatin modification combat data integration embryo tissue epigenetic regulation functional genomics genomic data hematopoietic differentiation high risk histone modification humanized mouse improved outcome in vivo individualized medicine insertion/deletion mutation leukemia leukemogenesis loss of function methylation pattern mouse model mutant new therapeutic target novel novel therapeutics nuclease pre-clinical premature progenitor programs protein protein interaction rational design stem targeted treatment therapeutic target therapy design transcription factor transcriptome transcriptome sequencing tumor vector whole genome

项目摘要

PROJECT SUMMARY/ABSTRACT Pediatric mixed phenotype acute leukemia (MPAL) is a rare, high-risk leukemia that accounts for 2-3% of pediatric leukemia cases and has a particularly poor prognosis (<50% survival). This is primarily due to lack of tailored therapies that target the unique genetic and developmental basis of this disease, highlighting a critical need to better understand MPAL biology. We recently discovered that the WT1 gene is mutated in nearly half of all pediatric T/myeloid MPAL patients, with most mutations causing premature termination codons that remove the C-terminal DNA binding domain of the WT1 protein. This particular alteration is predicted to have functional consequences, as WT1 encodes a DNA binding transcription factor important for early hematopoietic development. Furthermore, WT1 plays important roles in the regulation of gene expression, chromatin structure, and DNA methylation through interactions with other molecular complexes, highlighting the multiple pathways that may be disrupted in WT1-mutant hematopoietic cells. Importantly, each of these pathways represent potential vulnerabilities that can be exploited or directly targeted with rationally-designed therapy. The research proposed in this Fellowship will identify the developmental and molecular consequences of WT1 mutations in early hematopoiesis, providing a critical first step towards developing new strategies to combat this difficult to treat childhood disease. Aim 1 of this research proposal will address the role of truncating WT1 mutations in perturbing early hematopoiesis. A CRISPR/Cas9-based approach will be used to introduce patient-relevant mutations at the endogenous WT1 locus in human hematopoietic stem and progenitor cells (HSPCs) which will faithfully model the genetic and developmental origin of T/myeloid MPAL. Wild-type and WT1-mutant cells will be differentiated in vitro using colony forming assays and in vivo through transplantation into humanized mice. Flow cytometry and gene expression profiling will identify distinct hematopoietic populations and gene expression programs that are disrupted by WT1 alterations. Aim 2 of this research proposal will use proteomics and functional genomics assays to identify the molecular alterations induced by truncating WT1 mutations in human HSPCs, complementing the developmental alterations identified in Aim 1. Specifically, these assays will map dynamic protein-protein interactions, chromatin modifications, and DNA methylation patterns in wild-type and WT1-mutant HSPCs. Integration of these data will enable identification of the molecular mechanisms underlying transcriptional changes that deregulate early hematopoiesis in WT1-mutant MPAL. Overall, these studies will elucidate the developmental and molecular consequences of recurrent WT1 alterations in T/myeloid MPAL and reveal new pathways and dependencies that may be interrogated for therapeutic benefit.

项目概要/摘要小儿混合表型急性白血病 (MPAL) 是一种罕见的高危白血病，占儿童的 2-3% 儿童白血病病例的预后特别差（<50% 存活率）。这主要是由于缺乏针对这种疾病独特的遗传和发育基础的定制疗法，强调了关键的需要更好地了解 MPAL 生物学。我们最近发现近一半的WT1基因发生突变所有儿科 T/骨髓 MPAL 患者，大多数突变会导致过早终止密码子，从而消除 WT1 蛋白的 C 端 DNA 结合域。这种特殊的改变预计具有功能性后果，因为 WT1 编码对早期造血很重要的 DNA 结合转录因子发展。此外，WT1在基因表达、染色质结构、以及通过与其他分子复合物相互作用的 DNA 甲基化，突出了多种途径 WT1 突变的造血细胞中可能会被破坏。重要的是，每条途径都代表可以利用合理设计的治疗方法或直接针对的潜在漏洞。研究该奖学金中提出的建议将确定 WT1 突变的发育和分子后果早期造血，为制定新策略来对抗这种难以治疗的疾病提供了关键的第一步治疗儿童疾病。本研究提案的目标 1 将解决截断 WT1 突变在扰乱早期造血。基于 CRISPR/Cas9 的方法将用于引入与患者相关的人类造血干细胞和祖细胞 (HSPC) 内源性 WT1 位点的突变忠实地模拟 T/髓样 MPAL 的遗传和发育起源。野生型和 WT1 突变细胞使用集落形成试验在体外进行分化，并通过移植到人源化小鼠体内进行体内分化。流式细胞术和基因表达谱将识别不同的造血群体和基因被 WT1 改变破坏的表达程序。该研究计划的目标 2 将使用蛋白质组学和功能基因组学测定来鉴定截短 WT1 突变引起的分子改变人类 HSPC，补充了目标 1 中确定的发育改变。具体来说，这些测定将绘制野生型中动态蛋白质-蛋白质相互作用、染色质修饰和 DNA 甲基化模式和 WT1 突变 HSPC。这些数据的整合将能够识别分子机制 WT1 突变 MPAL 中早期造血失调的潜在转录变化。总体而言，这些研究将阐明 WT1 反复改变的发育和分子后果 T/骨髓 MPAL 并揭示可用于治疗的新途径和依赖性益处。